The principal focus of this project is to provide collaborating investigators with immunomodulators, conjugates and vaccine constructs that are specifically designed to answer well- formulated research questions in both basic and applied fields of immunology. This Section participates in planning the concepts of experiments, designs special constructs involving the linking of large molecules (macromolecules) of synthetic or natural origin to small molecular substances or other macromolecules, and synthesizes these constructs. Over the past year, our work in this project has mainly been confined to three collaborations that are described briefly below: 1. An HIV peptide conjugated to heat-killed Brucella abortus promotes anti-viral responses in immunodeficient mice. These studies are aimed at developing an effective immunotherapeutic vaccine for treating HIV-infected, immunocompromised individuals. As a model for such patients, Drs. B. Golding, H. Golding and D. E. Scott and their staff (CBER, FDA) have employed MHC class II deficient ("knockout") mice (II-/- mice), since these mice have low CD4+ T cell numbers, defective CD4-dependent (T-helper) responses, and are susceptible to opportunistic infections. We provided these investigators with conjugates of a synthetic 18- residue analog from the V3-loop structure of HIV-1 (MN), an immunodominant section of the virus envelope protein. The conjugate carriers were heat-killed bacteria from a strain of Brucella abortus (BA) and, for comparison, a typical T-dependent antigen, keyhole limpet hemocyanin (KLH). When injected into normal (WT) and II-/- mice, V3-BA but not V3-KLH induced high levels of cytokines, IL-12, IFNg (associated with a favorable Th1 type response), and IL-10 mRNA. In both WT and II-/- mice, V3- BA but not V3-KLH developed serum anti-V3 antibodies of IgA and predominantly IgG2a, IgG2b and IgG3 isotypes. Sera from both WT and II-/- mice inhibited syncytia development at titers similar to those observed from a non-progressing, HIV-1-infected subject, even though titers in the II-/- were significantly less than those in WT. Thus, V3-BA induced potentially therapeutic antibodies in these immunodeficient mice and a cytokine profile that supports a good cellular immunity. BA may therefore be a suitable carrier for inducing responses in HIV-1 infected persons who have diminished T-helper activity. 2. Studies on the effects of antigen epitope affinity and valence and of CD21 co-ligation on activation thresholds and activation-related apoptosis of human B cells. We have designed and prepared a number of antibody-dextran conjugates that have been employed by Dr. Patricia Mongini at NY University in experiments aimed at exploring how the manner of presentation of an antigen at the B-cell surface affects activation thresholds and the cellular and biochemical events that follow activation of the resting, mature cell. In a paper published this past year (see below), we reported that dextran conjugates, bearing both antibodies specific for the B-cell antigen receptor (anti-IgM as a surrogate, panclonal antigen) and complement receptor 2 (anti- CD21 or -CR2), can successfully co-ligate (co-cluster) these receptors at the B-cell surface. The findings suggest that the ability of a moderately multivalent antigen to engage B cells through both the antigen receptor (mIgM) and CD21 is critical for B-cell activation at limiting antigen concentrations and for eliciting initial immune responses in individuals in whom the majority of specific B cells are of low affinity for the antigen (see, Publications, below). In another series of experiments, using anti-IgM-dextran conjugates to activate B cells (two manuscripts in preparation), it was shown how antigen affinity and valence influences activation-related apoptosis at the G1 to S phase transition of the cell cycle. Also, evidence was gained for an upper (in addition to a lower) affinity threshold for anti-IgM-induced apoptosis in a human B lymphoma (Ramos) cell line. 3. A study of an unusual mechanism for ligand antagonism. We provided a library of nitrophenyl haptenic compounds and reagents (some synthesized anew for this study) to Drs. Torigoe and Metzger for exploring the following issue: Where a cascade of biochemical events follows ligand binding, the ratio of late to early events, stimulated by ligand binding to the mast cell, receptor-bound immunoglobulin (IgE), correlated directly with the ligand?s affinity. Moreover, low-affinity stimuli inhibited late responses to the high affinity ligand even when simple displacement of the competent ligand was precluded. A variety of experiments support the hypothesis that receptors clustered by the weakly binding ligand hoard a critical initiating kinase (e.g., lyn) thereby rendering incompetent the outnumbered clusters engendered by the high-affinity ligands. A similar mechanism could explain the antagonistic action of some (variant) peptides on the activation of T cells (Science, in press; see below).